Abstract Critical illness describes the life-threatening state caused by immune dysfunction that can occur following traumatic injury, shock, burn or major surgery. Trauma-induced immune incompetence enhances susceptibility to secondary infections that are a significant cause of mortality in hospital ICUs. Neutrophils, the most abundant leukocyte in the circulation, are critical for host defense against bacterial and fungal pathogens, but they also induce bystander tissue injury due to the non-specific and cytotoxic nature of their antimicrobial arsenal. The effects of trauma on neutrophils contribute to immune dysfunction and include a significant deficit in neutrophil capacity to find and fight infection, coupled with a primed phenotype associated with inducing tissue damage. The long-term goals of my research program are to understand mechanisms of neutrophil recruitment and effector function in health and disease. This MIRA application describes our synergistic approaches to identifying and understanding mechanisms of neutrophil pathobiology during critical illness. First, we have established an innovative approach to derive neutrophils ex vivo that circumvents the technical barriers to their genetic modification. This enables us to perform multiplexed forward genetic screening using CRISPR-Cas9 in neutrophils, and thereby identify new mechanistic aspects of their recruitment (e.g., integrin activation) and antimicrobial function (e.g., phagocytosis). Second, we have established a mouse model of critical illness by respiratory infection secondary to hemorrhagic shock. As the lungs are particularly susceptible to infection secondary to trauma and are also prone to neutrophil-mediated injury, this model provides a means to evaluate new targets/strategies for promoting selective aspects of neutrophil function to enhance host defense without exacerbating tissue damage. Finally, we will apply human trauma patient material (blood, plasma, lung fluid) to these approaches to probe disease-specific mechanisms of the neutrophil response. Since our studies will interrogate fundamental biological mechanisms, such as integrin activation, this work will also have broad impact on multiple fields. Together, these integrated approaches will address major knowledge gaps in neutrophil biology by spanning the molecular, cellular and organism/disease levels of experimentation.